Infectious disease is the second leading cause of death worldwide, the growing number of antibiotic-resistant microbes threatens to worsen this problem, but the pipeline for new antibiotics is running dry. The discovery of platensimycin (PTM) and platencin (PTN) as the third entirely new class of antibiotics in the past four decades is therefore of exceptional importance, representing the first step towards a new first-in-class antibacterial drug. However, development of PTM and PTN into commercial drugs faces formidable obstacles, including improvement of the drugs'pharmacokinetics and the inevitable emergence of resistance. In this application, we propose to: (i) characterize the PTM and PTN biosynthetic pathways, (ii) study the novel chemistry and enzymology for PTM and PTN biosynthesis, (iii) apply combinatorial biosynthesis methods to the PTM and PTN biosynthetic machinery to generate novel analogs, and (iv) investigate PTM and PTN self-resistance in Streptomyces platensis MA7327 (a PTM/PTN dual producer) and MA7339 (an exclusive PTN producer). Our hypotheses are: (i) studies of PTM and PTN biosynthesis promise to uncover new chemistry and enzymology because of their unprecedented molecular scaffolds, (ii) PTM and PTN share a common biosynthetic pathway, in which divergence leading to PTM and PTN is dictated by novel terpenoid synthases, (iii) application of combinatorial biosynthesis methods to the PTM and PTN biosynthetic machinery provides an outstanding opportunity to generate novel analogs, some of which could have improved therapeutic properties, and (iv) PTM and PTN self-resistance mechanisms in the producers serves as an excellent model to predict, understand, and combat future PTM or PTN resistance. The long-term goal of our research is to develop PTM and PTN as members of a new class of clinically useful antibacterial and antimalaria drugs. The specific aims for this grant period are: (i) functional characterization of the PTM and PTN biosynthetic machinery in vivo, (ii) biochemical characterization of the novel enzymes for PTM and PTN biosynthesis in vitro, (iii) application of combinatorial biosynthesis methods to PTM and PTN biosynthetic machinery for titer improvement and analog generation, and (iv) investigation of PTM and PTN self-resistance mechanisms in the producers. The outcomes of the proposed studies will include: (i) establishment of a unified biosynthetic pathway for the PTM and PTN class of natural products, (ii) discovery of novel chemistry and enzymology for aminobenzoate and terpenoid biosynthesis, (iii) production of PTM and PTN analogs for SAR (structure-and-activity-relationship) evaluation as antibacterial drugs, and (iv) understanding of PTM and PTN self-resistance mechanisms in the producers. PUBLIC HEALTH RELEVANCE: Infectious diseases and antibiotic resistance pose serious threats to public health, and ensuring the continued availability of novel antimicrobials to combat existing and emerging pathogens, especially pathogens expressing resistance to currently available therapies, is a critical public health issue. Platensimycin (PTM) and platencin (PTN), as the third entirely new class of antibiotics discovered in the past four decades, represent the first step towards a new first-in-class antibacterial drug. However, development of PTM and PTN into commercial drugs faces formidable obstacles. This research will study PTM and PTN biosynthesis and resistance and to produce PTM and PTN analogs for structure-and-activity relationship studies. The outcomes include development of PTM and PTN into a new class of clinically useful antibacterial drugs and understanding of PTM and PTN resistance mechanisms in their native producers to predict and combat future drug resistance in the clinic.